Abstract

The Civilian Control Zone (CCZ) in South Korea is a highly restricted area with significant ecological value due to limited human access. This study explores the feasibility of non-invasive biodiversity monitoring using spider webs as environmental DNA (eDNA) collectors in the CCZ, particularly where traditional field surveys are constrained. A total of six spider webs were collected along the DMZ Peace Trail in Hwacheon-gun, Gangwon province, and subjected to eDNA metabarcoding using vertebrate-specific primers. Despite quality limitations in Q20 and Q30 scores, over two million reads were generated, and 531,429 high-quality reads were retained after filtering with the DADA2 pipeline. Taxonomic assignment using BLAST identified 13 vertebrate species, including nine mammals, two amphibians, and two birds. Comparative analysis with conventional field surveys revealed limited species overlap, but spider web eDNA successfully detected cryptic or rarely observed species, such as Rattus norvegicus and Felis catus. These findings suggest that spider web-derived eDNA offers potential as a complementary tool, especially in areas where direct observation is difficult or impossible due to safety or security concerns. However, incomplete reference databases and low detection efficiency highlight the need for improvements in sampling methods and local DNA libraries. This study presents a promising step toward developing alternative, non-invasive ecological monitoring techniques applicable to inaccessible terrestrial environments.